臺灣博碩士論文加值系統

在過去的研究中假設抑癌基因 (Tumor suppressor genes) 或二維基因座 (Bivalent loci)異常的去氧核醣核酸甲基化 (DNA methylation) 會導致癌症，因此我的研究主要是: 辨認關鍵的抑癌基因或二維基因座的甲基化是否對於乳癌的發生是必須的。從79對乳癌病人檢體中，我們發現在乳癌病人檢體中的腫瘤區域和相鄰的正常區域中，抑癌基因RassF1A異常甲基化情形有顯著差異。另外，二維基因座ENSA的甲基化也在早期與晚期的乳癌中有顯著差異。接著將表達RassF1A及HIC1之質體轉染至MDA-MB-231及MCF7細胞中，以聚合酶連鎖反應及西方點墨法驗證，並以免疫染色螢光觀察過度表達之細胞，另外透過MTT assay發現在MDA-MB-231細胞中RassF1A及HIC1過度表達會使細胞生長速率下降，在MCF7細胞中則無此情形。

Abnormal DNA methylation within tumor suppressor (TS) genes and/or bivalent loci was hypothesized to cause cancer. Therefore, we aimed to identify the crucial TS or bivalent loci that their methylation might be sufficient for the development of breast cancer. From 79 pairs of breast cancer samples, we found significant RassF1A methylation differences between the tumor and adjacent normal parts. On the other hand, the abnormal ENSA methylation was found to correlate with low and high stage breast cancer progression. At last, overexpressed RassF1A and HIC1 in breast cancer cell lines, MDA-MB-231 and MCF7, was validated by immunostaining and found to only reduce the cell proliferation rate in MDA-MB-231.

謝辭 I
中文摘要 II
英文摘要 III
目錄 IV
圖表目錄 VII
第一章 緒論
第一節 乳癌 (Breast Cancer) 1
第二節 癌症對位性基因體學 (Cancer Epigenetics) 2
第三節 去氧核醣核酸甲基化 (DNA Methylation) 3
第四節Endosulfine alpha (ENSA) 5
第五節 目的 6
第二章 材料與方法
第一節 染色體去氧核醣核酸萃取 8
壹、 細胞染色體去氧核醣核酸萃取 8
貳、病人檢體染色體去氧核醣核酸萃取 9
第二節 亞硫酸氫鹽轉換法 9
第三節 即時半定量甲基化特異性聚合酶連鎖反應 10
第四節 數據分析 11
第五節 細胞培養 12
第六節 細胞轉染 12
第七節 篩選穩定細胞株及驗證 13
第八節 核糖核酸萃取 14
第九節 反轉錄反應 14
第十節 即時定量聚合酶連鎖反應 15
第十一節 西方點墨法 16
壹、蛋白質萃取 16
貳、蛋白質濃度定量 16
參、聚丙烯醯胺膠體製備與電泳 17
肆、轉漬 18
伍、冷光呈色顯影 19
第十二節 免疫螢光染色 19
第十三節 細胞生長速率分析 20
第三章 實驗結果
第一節 乳癌檢體中多櫛家族蛋白質基因座及抑癌基因座甲基化狀態 22
第二節 多櫛家族蛋白質基因座與抑癌基因座在乳癌檢體中的甲基化狀態可區分成兩類 23
第三節 特定基因的甲基化與乳癌檢體的臨床病理特徵相關 23
第四節 在乳癌細胞株中表達RassF1A和HIC1 24
第四章 討論 26
參考文獻 28
圖表 36

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